The most commonly used methods for the quantification of gene expression include northern blotting (Watson, J., et al., Recombinant DNA, 2nd Edn. W.H. Freeman and Company, New York, 1992), ribonuclease protection (Chan, S. D. H, et al., Anal. Biochem., 242, 214, 1996) and reverse transcription-polymerase chain reaction (RT-PCR)(Cottrez, F., et al., Nucleic Acids Res., 22, 2712, 1994; Totze, G., et al., Mol. Cell. Probes, 10, 427, 1996). The first two methods require 10-100 μg of mRNA and can detect single mRNA molecules at 106-107 copy levels. Such quantities can be easily isolated from bulk tissues, but if one has to quantify a number of genes in limited amounts of sample or has a need to separate only certain types of cells for analysis, northern blotting and ribonuclease protection techniques are not feasible. RT-PCR can theoretically amplify a single nucleic acid molecule by millions of times and thus could be very useful for very small sample sizes. However, RT-PCR amplification tends to introduce contamination. It also requires considerable optimization of primer sets and sample pretreatments, therefore, prolonging assay time. More frequently, different amounts of mRNA sequences in a starting mRNA mixture may not be represented at the same level in the final RT-PCR products due to selective and non-linear target amplifications. Incomplete denaturation of RNA secondary structure during the cDNA synthesis step can also halt the polymerase, resulting in shorter cDNA copies of the target mRNA. These limitations affect the precision and quality of the resulting data, and often provide distorted information of gene expression. Multiple replicates can help to gain confidence in the results for such experiments, but that is not applicable to small or rare samples.
To circumvent the above-mentioned problems associated with RT-PCR, techniques that use a cisplatin-digoxigenin derivative conjugate to directly label nucleic acid molecules have recently been developed (Hoevel T., et al, Biotechniques, 27(5):1064-7, 1999). The key advantage of the direct nucleic acid labeling methodology is that it is simple, fast and less perturbing to the nucleic acid molecules. The resulting labeled nucleic acid allows a greater accuracy in the identification of differentially expressed genes. However, the quantification of gene expression has proven to be difficult owing to the limited sensitivity of the existing nucleic acid detection techniques.
Usually micrograms of mRNA is needed for quantitative purposes (Hoevel T., et al., Biotechniques, 27(5):1064, 1999; Boon, E. M., et al., Nat. Biotechnol., 18, 1096, 2000). Sensitive gene detection is one of the challenges in current and future molecular diagnostics.
Recent advances in developing bioelectronic DNA analysis systems open up new opportunities for molecular diagnostics and have attracted substantial research efforts (Boon, E. M., et al., Nat. Biotechnol., 18, 1096, 2000; Rodriguez, M. & Bard, A. J. Anal. Chem., 62, 1658, 1990). Optical (Jordan, C. E., et al., Anal. Chem., 69, 4939, 1997; Fotin, A. V., et al., Nucleic Acids Res., 26, 1515, 1998), electrochemical (Kelley, S. O., et al., Bioconjug. Chem., 8, 31, 1997; Kelly, S. O., et al., Nucleic Acids Res., 27, 4830, 1999), and microgravimetric and quartz-crystal microbalance (Bardea, A., et al., Chem. Commun., 839, 1998; Wang, J., Nucleic Acids Res., 28, 3011, 2000), transduction methods have been reported for the detection of DNA hybridization events. Amplified electronic transduction of nucleic acid recognition events (Caruana, D. J. and Heller, A., J. Am. Chem. Soc., 121, 769, 1999; Patolsky, F., et al., Chem. Int., 40, 2261, Ed. 2001; Patolsky, F., et al., J. Am. Chem. Soc., 122, 418, 2000; Zhang, Y., et al., Anal. Chem., 75, 3267, 2003) has also been addressed recently. The inherent miniaturization of electrochemical biosensors and their compatibility with advanced semiconductor technologies promise to provide a simple, accurate and inexpensive platform for an early diagnosis of genetic diseases. Despite the enormous progress made in electrochemical nucleic acid biosensors in the past 5 years, in order to be one step closer to the market several important hurdles need to be overcome. The first is to test the biosensors on genomic nucleic acid from real-world samples (Lay, P. A., et al., Inorg. Synth., 24, 291, 1986). So far, most of the electrochemical biosensors start with relatively short synthetic oligonucleotides, or with a round of PCR amplification. Another challenge is to multiplex the electrochemical biosensors and their fabrication into useful sensor arrays. Typically, arrays of 30 to 100 are needed for diagnostic purposes. For example, breast cancer screening requires testing for 20-30 cancer marker genes in addition to positive and negative controls (Drummond, T. G., et al., Nat. Biotechnol., 21, 1192, 2003).
Accordingly, there is a need in the art for the development of improved and efficient methods for the identification and/or analysis of small amounts of nucleic acids. In particular, there is a need for improved and efficient methods for the direct identification and/or analysis of target genes in the total mRNA present in a sample.